It has recently been shown in animal and human studies that thymidine (TdR) administration offers significant protection against methotrexate (MTX)-induced host toxicity with retained antitumor effects. Other work in cell culture systems suggests that MTX can cause cytotoxicity, not overcome by exogenous TdR, due to inhibition of purine biosynthesis. We now propose to investigate methods for improving the therapeutic efficacy of MTX by using exogenously supplied thymidylate precursors in vivo utilizing model animal tumor systems (mouse leukemias and rat colon-derived adenocarcinoma). The relative dependency of host and tumor tissues on thymidylate versus purine biosynthesis will be explored using radiolabeled nucleic acid precursors, especially (14C) formate. Studies will be undertaken to determine whether thymine (T), which lacks the deoxyribose moiety of TdR, might be a more selective host protective agent than TdR. Since T catabolism occurs to a greater degree in normal tissues and inhibition of catabolism by agents such as diazouracil tends to selectively increase incorporation of T into normal tissues, appropriate combinations of T or TdR plus diazouracil might increase selective antitumor effects with MTX. A major emphasis of the work we propose is to determine whether resistance developing under treatment with halogenated pyrimidines, especially fluorodeoxyuridine (FUdR) and fluorouracil (FU), induces collateral sensitivity to MTX/TdR or MTX/T therapy due to loss of ability to transport or activate exogenous TdR or T to thymidylate (TMP). Resistant tumor sublines will be developed and the mechanisms of resistance explored using radiolabeled precursor studies in vivo and in vitro. Dose-scheduling studies will then be performed to determine how to maximally capitalize on the collateral sensitivity produced.